Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

ABSTRACT Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

MAbs, 242-3 and 270-12, had similar sequences with an amino acid identity of 99% and similar epitope recognition in the lateral ridge of domain III. SHM/FWR mutation rates (MAb 242-3, 12.1%/6.3%; MAb 270-12, 11.1%/5.1%) and CDR3 lengths (17 amino acids for both) were intermediate between MAbs 289-3 and 306-2. Interestingly, the ZIKV prM-specific non-neutralizing MAb 278-11 also had a high SHM/FWR mutation rate of 17.9%/11.5% and a high CDRH1,2 mutation rate of 47.1%. The CDR3 length of MAb 278-11 was 12 amino acids. In conclusion, among ZIKV-specific neutralizing MAbs, we observed higher somatic mutation and the longest CDR3 length in the MAb recognizing a quaternary epitope. There was no clear trend between epitope mapping information and SHM, FWR mutation, and CDR information for the light-chain variable region, except that the longest CDR3 length (16 amino acids) was also observed in MAb 289-3, which recognizes a quaternary epitope ( Fig. 4B and Table 3).
Impact of framework amino acids on the binding activity of anti-ZIKV MAbs. To understand the impact of the FWR mutations on MAb binding, all FWR amino acids of anti-ZIKV domains I to III and MAb 102-1, 270-12, 289-3, and 306-2 were reverted to the germ line amino acids of the allele and characterized (Fig. 6, Table 5). Reversion of 4 FWR heavy-chain (FWRH) and 12 FWR light-chain (FWRL) amino acids in MAb 270-12 resulted in the loss of binding activity to ZIKV E protein and reduced binding to ZIKV-VLPs ( Fig. 6B and F). Reversion of 9/9 and 3/5 amino acids of FWRH/FWRL chains of MAb 102-1 and 306-2, respectively, increased the dissociation rate of ZIKV E protein binding. However, there were no differences in binding to ZIKV-VLPs (Fig. 6A, D, E, and H). Although the highest rate of mutation of FWR H and L chains was observed in MAb 289-3, reversion of 13 FWRH and 9 FWRL amino acids of MAb 289-3 did not alter binding to either ZIKV E protein or ZIKV-VLPs ( Fig. 6C and G).

DISCUSSION
We identified and characterized eight unique ZIKV-specific rabbit MAbs with diverse qualities, including epitope specificity, neutralizing activity, and degree of affinity maturation. Rabbits represent an alternative species to generate MAbs with properties similar to those of human MAbs. Rabbits are evolutionarily distinct from mice and other rodents, and rabbit and rodent antibody ontogeny also differ (36). Rabbit antibodies have a long average CDRH3 of 14.8 6 3.6 amino acids, which is similar to the average human  (39)(40)(41)(42). The CDRH3 length of the rabbit anti-ZIKV neutralizing MAbs recognizing conformational epitopes described here had 12 to 18 CDRH3 amino acids. Rabbit immunoglobulin genes also undergo a high degree of variable region rearrangement (39). The SHM rates of the identified rabbit anti-ZIKV MAb genes ranged from 5.4% to 10.5%, compared to SHM rates of published human and mouse anti-ZIKV MAbs of 2.7% to 10.4% (43,44). Potently neutralizing ZIKV-specific human MAbs have been described that map to the domain III lateral ridge (43,(45)(46)(47), domain II (47,48), or to complex epitopes spanning multiple domains (49,50), while fusion loop-specific MAbs are more likely to be cross-reactive with DENV (42). Three of the rabbit MAbs described here map to the domain III lateral ridge, a region that is also targeted by several mouse and human MAbs that have demonstrated ZIKV-neutralizing activity and protective immunity in mouse models, suggesting that this is an immunodominant region for ZIKV-specific neutralizing antibodies in multiple species (45,47,48,51). The epitopes recognized by MAbs 102-1 and 270-12 include residue S368 in the domain III lateral ridge, which has been determined to be an important residue for human ZIKV-specific neutralizing antibodies (52). MAb 242-3 also recognized the domain III lateral ridge, but while S368 was not identified as a critical residue, the adjacent residues T369 and E370 were identified as critical. Other human ZIKV-specific neutralizing antibodies, including ZIKV-116, 7B3, and ZK2B10, recognize domain III lateral ridge epitopes (47, 48, 53) whose residues are overlapping but distinct from those of the rabbit MAbs described here. These results suggest that domain III immunodominant ZIKV-specific epitopes recognized by neutralizing rabbit MAbs are similar to epitopes recognized by human ZIKV-specific MAbs, with the exception of ZIKV-specific neutralizing MAb 306-2, which recognizes a novel conformational epitope at the distal end of domain III. Critical residues (green spheres) for antibody binding are visualized on a crystal structure of the ZIKV E protein dimer (PDB entry 5IRE, 73) or on a cryoelectron microscopy structure of ZIKV precursor membrane prM protein for 278-11 (PDB entry 5U4W, 74). Secondary residues (gray spheres) that may contribute to binding are also shown. Red, E protein domain I; yellow, domain II; blue, domain III. Detailed data are shown in Table 2.
Potently neutralizing antibodies recognizing complex and quaternary epitopes have been described for a number of viruses, including ZIKV, DENV, and HIV (44,49,54,55). Among the MAbs described here, MAb 289-3 had the strongest neutralizing activity and recognized a quaternary epitope, including critical amino acids in both domains I and III. Previously, a rationally engineered MAb designed to target a quaternary epitope spanning an epitope proximal to the fusion loop was capable of broadly neutralizing ZIKV strains and conferred protection against vertical transmission and fetal mortality in mice (49). Modeling studies suggest that MAbs targeting this region constrain the E protein structure and block fusion (49). Further studies will be required to determine the structure of MAb 289-3 complexed with ZIKV E protein. Three ZIKV-specific human MAbs have recently been described that also span an epitope in domains I and III (44,50). Alanine-scanning mutagenesis identified the critical residues recognized by two of these MAbs, B11F and A9E, as mapping within domain I alone. Two critical domain I residues of MAb 289-3, E162 and G182, are described as escape mutation sites for MAb A9E (50,56). We also note that the critical residues K301 (domain III) and G182 (domain I) were also identified by shotgun mutagenesis analysis as critical for binding by the third MAb, protective anti-ZIKV MAb MZ4, which binds a site centered on the E protein domain I/III linker region (44).
CDRH3 length was associated with increased neutralizing antibody activity. A high degree of SHM and relatively long CDRH3 has been associated with the evolution of potent neutralization activity as well as with recognition of complex quaternary epitopes (57). Among the MAbs with ZIKV neutralizing activity, the strength of binding was associated with higher heavy-chain SHM, CDRH, and FWR mutation rates. Our findings were consistent with those for anti-ZIKV EDE1 MAbs C8 and C10, which bind across E protein dimers to strongly neutralize ZIKV (58) and show a high rate of heavychain gene SHM, 6.9% and 2.8%, and longer CDRH3 length, 15 and 21 amino acid residues, respectively (42). The association between somatic mutation rate and increased antibody affinity is well established (28,59,60). Characterization of MAb 289-3 demonstrates that a high degree of SHM and long CDRH3 can be achieved by ZIKV vaccination and can lead to the evolution of antibodies with potent ZIKV-specific neutralizing activity. As expected, strong correlations were observed between antibody binding parameters and heavy-chain CDR mutation rate, since CDRs make up the antigen-binding site. Strong correlations were also observed between antibody binding parameters and FWR mutation rate, which was less expected as FWRs likely do not directly bind but provide structural support for the CDRs. FWR mutations may increase antibody flexibility, facilitating CDR contact with epitopes (33,34). The role of FWR mutations in potency and neutralization of anti-HIV MAbs is variable depending on the specific antibody (61). FWR mutations are important for MAbs against anti-vascular endothelial growth factor, VEGF (34), and FWR mutations have been widely applied, stabilizing the structure of humanized MAbs derived from mice (62). We found that binding activity of MAbs 102-1, 306-2, and 270-12 was impacted by reversion of FWR mutations but that FWR mutations were not essential for binding activity of the most potent MAb, 289-3. This result for MAb 289-3 was different from our expectations, since eight amino acids, the highest number among four MAbs, were changed in heavychain FWR3 regions supporting CDR3, and the threonine residue at position 92 (in the international ImMunoGeneTics information system, termed IMGT numbering) (63) was mutated to proline. Although the introduction of a proline residue might be thought likely to perturb the FWR structure, a proline at position 61 was critical for the thermal stability of a broadly neutralizing anti-HIV MAb 3BNC60 (32). Further studies are required to more fully understand the role of FWR amino acids in anti-flavivirus MAb specificity and activity.
In summary, we discovered eight ZIKV-specific MAbs against distinct regions of envelope and prM proteins, including a potent neutralizing MAb that recognized a quaternary epitope spanning domains I and III and a non-neutralizing MAb that recognized a linear epitope on the ZIKV prM protein. Detailed characterization of the rabbit MAbs demonstrated that ZIKV-specific MAbs recognizing conformational and quaternary epitopes on the ZIKV E protein bind with high affinity and are neutralizing. There were significant correlations between the SHM rate, FWR mutation rate, and antibody binding parameters. The higher degree of CDR mutation and SHM, and longest CDRH3, were found in a MAb recognizing a quaternary epitope spanning ZIKV E domains I and III. For some MAbs, reversion of FWR mutations to the germ line allele reduced the affinity of antigen-binding. Thus, we conclude that both SHM and FWR mutations of anti-ZIKV MAbs contribute to antibody affinity, specificity, and functionality.

MATERIALS AND METHODS
Ethics. All procedures were conducted in compliance with the U.S. Department of Agriculture's Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals (64), and the National Institutes of Health, Office of Laboratory Animal Welfare. Whenever possible, procedures in this study were designed to avoid or minimize discomfort, distress, and pain to animals. The animal immunization experiment protocols were approved by the IACUC (International Animal Care and Use Committee) at LabCorp (Denver, PA, USA).
Antigens and other reagents. ZIKV (strain; PRVABC59; CDC, Fort Collins, CO) was grown in Vero cells, harvested, purified, and formalin inactivated. These purified inactivated Zika viruses (PIZV) were formulated with aluminum hydroxide. DENV Rabbit immunization and spleen cell preparation. Two New Zealand white female rabbits (LabCorp, Denver, PA, USA) were immunized intramuscularly (i.m.) with 5 mg of PIZV plus aluminum hydroxide on days 0, 14, 28, 56, and 95. Both rabbits were boosted i.m. with 5 mg ZIKV-VLP in Freund's incomplete adjuvant on day 109, followed by intravenous injection of 5 mg ZIKV-VLP on day 130. Splenocytes from rabbits were isolated 4 days after the final boost. The spleen cells were dispersed and subjected to red cell lysis. The cells were frozen in a freezing medium (90% fetal bovine serum and 10% dimethyl sulfoxide) in liquid nitrogen.
Anti-ZIKV MAb hybridoma generation and clone selection. Eight hundred million rabbit splenocytes were fused with 400 million fusion partner cells (240E-W2 cells) (66) and plated into 80 96well plates. The hybridomas were cultured at 37°C, 5% CO 2 . After 14 days, 7,680 multiclonal supernatants were screened by enzyme-linked immunosorbent assay (ELISA) using ZIKV-VLP and ZIKV E protein. A total of 384 clones were positive for ZIKV-VLP alone, and 19 positive multiclones were  Table 3). Blue bar, MAb epitopes, ZIKV E protein domain III or domain I to III; green bar, MAb epitope, ZIKV E protein fusion loop (FL); yellow bar, MAb epitope, ZIKV precursor membrane (prM) protein. Boldfaced amino acid residues, critical amino acid of ZIKV E protein and prM protein for anti-ZIKV MAb binding (Table 2).

Somatic Hypermutation and Anti-ZIKV MAb Functions
Journal of Virology selected by both ZIKV-VLP and E protein. These multiclones were subcloned by limited dilution, and 155 submonoclones were determined by MAb production; ZIKV neutralizing activity; ELISA against DENV1-4 inactivated virus, ZIKV-VLP, and ZIKV E protein; and k dis ranking against ZIKV-VLP using Octet-96 Red (Sartorius, Fremont, CA, USA). We selected 14 clones with high antibody expression for further characterization, nine clones with neutralizing activity and five clones without neutralizing activity. DNA sequence analysis of anti-ZIKV MAbs. Hybridoma cells were collected and lysed for poly(A) 1 mRNA isolation using poly(A) 1 RNA isolation kit. Reverse transcription-PCR was conducted using RNA products and synthesized cDNA. First, the rabbit IgG variable region of heavy-chain and full-length lightchain were individually PCR amplified using gene-specific primers. Following gel purification of PCR products, the entire light-chain fragment was cloned into a mammalian light-chain expression vector. Next, the heavy-chain variable fragment was fused with rabbit heavy-chain constant region expression vectors.
Anti-ZIKV MAb expression and purification. To express recombinant rabbit monoclonal antibodies, the light-and heavy-chain mammalian expression plasmids were cotransfected into exponentially growing 293-6E cells using lipid-mediated transfection reagent (67). The serum-free culture supernatant was harvested 5 days after transfection by centrifugation. Harvested culture medium was centrifuged to remove cell debris, and the clear supernatant containing secreted monoclonal antibodies was purified through MabSelect SuRe protein A column chromatography (Cytiva, Marlborough, MA, USA). The eluted antibody was dialyzed in phosphate-buffered saline (PBS) buffer, sterile filtered, and adjusted to pH 7.4.
Antibody expression and purification of anti-ZIKV allele reverted MAbs. The light and heavy chains of rabbit MAb mammalian expression plasmids were cotransfected into Expi 293 cells systems (Thermo Fisher, Waltham, MA, USA) (68), and the transfected medium was harvested 5 days after transfection with centrifuging. Monoclonal antibodies were purified through protein A Sepharose (Cytiva, Allele analysis. Anti-ZIKV MAb allele and CDR3 regions were analyzed by IMGT/V-QUEST (http:// www.imgt.org/IMGT_vquest/analysis) and NCBI IGBLAST (https://www.ncbi.nlm.nih.gov/igblast/). SMH rate, FWR mutation, and CDR mutation were calculated by mutated DNA and proteins in the variable region from the allele sequence.
Neutralization assay. A 50% tissue culture infective dose (TCID 50 )-based microneutralization test (MNT) was used for the virus-neutralizing activity of MAbs in 96-well plates. ZIKV (PRVABC59; CDC, Fort Collins, CO) grown in Vero cells was used as the challenge virus in the neutralization assay. First, hybridoma supernatants or diluted purified MAbs were incubated with 100 TCID 50 /well of ZIKV for 1.5 h at 37°C 5% CO 2 . Next, the ZIKV-MAb mixture was added to Vero cell monolayers in 96-well plates. The plates were incubated at 37°C 5% CO 2 for 5 days, and cytopathic effect was scored under light microscopy. Relative infectivity was plotted against MAb concentration, and IC 50 values were determined as described previously (46).
Western analysis. Western blot analysis was conducted by a capillary-based electrophoresis system (69) (Wes; ProteinSimple, Santa Clara, CA, USA). In brief, 27 or 240 ng ZIKV-VLP was denatured at 70°C without reducing agent for 5 min and loaded on a Wes assay plate and electrophoresed. Next, 10 mg/ mL anti-ZIKV MAb was charged, followed by Wes horseradish peroxidase-conjugated anti-rabbit secondary antibody. The sample run was analyzed by examining the electropherogram and digital gel image.
K D measurement. Antibody kinetic analyses were conducted by Octet HTX systems (Sartorius, Fremont, CA, USA). Briefly, 0.1 to 0.3 mg/mL anti-ZIKV MAb was conjugated to an amine-reactive 2nd generation (AR2G) biosensor (Sartorius, Fremont, CA, USA) using EDC/NHS at pH 4.0 or 5.0 in acetic buffer. A volume of 0.1 to 1.0 mg/mL ZIKV-VLP or 0.2 to 2 mg/mL ZIKV E protein in 1Â kinetic buffer (Sartorius, Fremont, CA, USA) was associated with anti-ZIKV MAb for 900 s and dissociated for 1,200 s. Kinetic parameters, association constant (k a ), and dissociation constant (k dis ) were analyzed by Octet Data Analysis Software HT (ver. 11.1.2.48 Sartorius, Fremont, CA, USA) with the Langmuir 1:1 binding model. Equilibrium dissociation constants (K D ) were calculated the following equation: Shotgun mutagenesis epitope mapping. Epitope mapping by shotgun mutagenesis and alaninescanning mutagenesis (71) was performed as described previously (47). A ZIKV (ZIKV SPH2015) prM/E alanine scanning mutation library was created, individually changing residues to alanine (or alanine residues to serine). A total of 672 ZIKV prM/E mutants (100% coverage of prM/E) were generated and transfected into HEK-293T cells. Cells were fixed in 4% (vol/vol) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and permeabilized with 0.1% (wt/vol) saponin in D-PBS plus calcium and magnesium (D-PBS11) before incubation with MAbs diluted in D-PBS11, 10% normal goat serum, and 0.1% saponin. Antibodies were detected using 3.75 mg/mL AlexaFluor488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with D-PBS11 and 0.1% saponin followed by two washes in D-PBS, and mean cellular fluorescence was detected using a high-throughput iQue flow cytometer (Sartorius, Fremont, CA, USA). MAb reactivities against each mutant prM/E clone were calculated relative to wild-type prM/E reactivity by subtracting the signal from mock-transfected controls and normalizing the wild-type prM/E-transfected controls. The counterscreen strategy facilitates the exclusion of mutants locally misfolded or has an expression defect (72).
Association/dissociation analysis of FWR mutation reverted anti-ZIKV MAb. Evaluation of anti-ZIKV MAb allele mutation reverted MAb was conducted by Octet HTX (Sartorius, Fremont, CA, USA). Briefly, anti-ZIKV MAbs were diluted to 2 mg/mL in 0.1% BSA-PBST buffer and captured by protein G biosensor (Sartorius, Fremont, CA, USA); 3 mg/mL ZIKV E protein was associated for 600 s and dissociated for 900 s in the same buffer.
MAbs preparation, Hetal Patel for leading ZIKV project, Jonathan Hernandez for data integrity review, Tawnya Nead and Mark Lyons for potency assay using anti-ZIKV MAbs, Eric Shaw for reviewing the draft manuscript, and Ralph Braun for suggestions and advice during the studies. We also appreciate researchers of Discovery Research Takeda Vaccine Business Unit for discussion and advice. In addition, LabCorp Antibody Reagents and Vaccines team immunized rabbits, and Abcam Antibody Discovery and Development Service provided support to generate anti-ZIKV MAbs. I